The long-term objective of this Program is to determine the fundamental mechanisms underlying the interplay between transcription factors, chromatin structure, and higher-order genomic organization during the cellular conversion to and maintenance of pluripotency. Despite the remarkable ability of transcription factors and miRNAs to convert cells to pluripotency, undefined stochastic parameters presently limit the efficiency of the process. In addition, different pluripotent lines have different capacities for terminal differentiation and we poorly understand parameters that determine how well in vitro differentiation compares to in vivo differentiation. Understanding the molecular mechanisms in pluripotency induction and maintenance as well as those limiting differentiation will allow enhancements of the process that, in turn, will facilitate the use of small human biopsy samples much more efficiently than present techniques allow. To this end, the four projects of the Program ask: 1) How is the differentiated cell genome reorganized within the nucleus, during reprogramming to pluripotency, what aspects of reorganization are important, and what controls genome organization in pluripotent cells? 2) How do ectopic pluripotency transcription factors gain access to silent, chromatinized target sites to activate the endogenous pluripotency network, and how can the process be enhanced? 3) What regulatory circuits need to be properly established within pluripotent cells to allow their subsequent differentiation to fully mature progeny? 4) What marks of the competence to differentiate exist in pluripotent cells and how do they get established? By seeking answers to these questions in a single Program, we can obtain a time-resolved, integrated view of the mechanisms by which different aspects of the nuclear genome change coordinately to properly convert a cell to pluripotency and the process by which cells return to the somatic state. We also anticipate that the coordinate mechanisms unveiled by our studies will provide insights into direct cell reprogramming, independent of pluripotency. Administrative and Bioinformatics Cores and a shared Web site will support the projects with integrated services for optimal quality, efficiency, and data-sharing. The Administrative Core leverages existing high throughput sequencing, microarray, and stem cell cores at the respective institutions. The Project and Core leaders have complementary expertise in the relevant areas of stem cell biology, differentiation, transcription and chromatin/ epigenetics and have a long-standing record of interactive collaborations and publications. The plan provides unique experimental synergies that address the objectives of the funding announcement.